Pilot Abstract The clinical course of cystic fibrosis (CF) is characterized by cycles of airway inflammation triggered by bacterial pathogens such as Pseudomonas aeruginosa and other Gram negative rod (GNR) organisms, leading to acute airway exacerbations and increased airway obstruction due to mucus plugging. Reducing the burden of bacteria in the airways by physical clearance and treatment with antibiotics remains the cornerstone of management of CF lung disease. The cumulative effects of multiple courses of antibiotics inevitably leads to multi-drug resistant (MDR) organisms. Thus, it is important to identify new therapeutic approaches that target GNR organisms in CF to reduce selective pressure that leads to MDR organisms and to have more treatment options once MDR organisms have developed. The current recommendation is that children colonized with P. aeruginosa receive azithromycin three time a week as this therapy has been shown to be anti-inflammatory and reduce pulmonary exacerbations. P. aeruginosa and other GNRs are non-susceptible to azithromycin in assays that are performed in traditional bacteriologic media. Thus, one would conclude that azithromycin does not have a direct antimicrobial effect on P. aeruginosa. However, recent data from Lin and Nizet et al (data generated from initial UC San Diego RPDP Center grant support) suggest that azithromycin may have direct antimicrobial effects in vivo in a murine model and could be used to treat highly resistant GNRs. Thus, in this pilot study we plan to harness state-of-the-art capabilities of the Knight and Dorrestein labs to better understand the microbiome and metabolomic changes of a short five day course of daily azithromycin in CF patients recently colonized with P. aeruginosa. This is key to understanding the future utility of azithromycin in decreasing the development of MDR infections and as a potent anti-microbial agent against MDR GNR infections. This will be achieved with the following Specific Aims: Specific Aim 1 will determine the microbiologic effects of daily azithromycin in CF subjects with P. aeruginosa colonizationby evaluating the in vitro sensitivities of P. aeruginosa respiratory isolates measured in eukaryotic cell culture media, and the respiratory microbiome and microbial metabolites in the Knight lab. Specific Aim 2willdetermine the metabolomic effects of azithromycin in CF subjects by evaluating the change in inflammatory markers as well as determining the relationship between drug level and anti-inflammatory response. This success of this pilot project is assured given the highly successful team that has been assembled: the strong new investigator, Dr. Kathryn Akong, a CF specialist; Dr. John Bradley, a pediatric infectious disease specialist with expertise in antimicrobial therapies as well as clinical trials and FDA regulatory approval of pediatric therapies; Dr. George Sakoulas, an adult infectious disease expert in the Nizet lab who was involved in the original in vivo mouse model experiments of azithromycin and GNRs; and Drs. Rob Knight and Pieter Dorrestein, world-renowned experts in the field of microbiome and metabolomics, respectively.